Reaction-based small-molecule fluorescent probes for chemoselective bioimaging

Abstract

The dynamic chemical diversity of elements, ions and molecules that form the basis of life offers both a challenge and an opportunity for study. Small-molecule fluorescent probes can make use of selective, bioorthogonal chemistries to report on specific analytes in cells and in more complex biological specimens. These probes offer powerful reagents to interrogate the physiology and pathology of reactive chemical species in their native environments with minimal perturbation to living systems. This Review presents a survey of tools and tactics for using such probes to detect biologically important chemical analytes. We highlight design criteria for effective chemical tools for use in biological applications as well as gaps for future exploration.

Figure 1. Overview of organic and metal-mediated reaction-based strategies for the chemoselective bioimaging of small-molecule and metal ion analytes in biological systems

Representative approaches to turn-on or ratiometric fluorescence detection by a, bond-cleavage reactions, b, organic addition and/or metal–ligand substitution reactions, and c, tandem reaction cascades to unmask a fluorogenic scaffold. The green ‘polyaromatic’ shape represents a generic fluorophore. d, A representative set of common dyes that can be transformed into fluorescent probes for chemoselective bioimaging, sorted by structure and emission colour.

Figure 2. Representative oxidative cycloaddition reactions for fluorescence bioimaging

a, Transformation of a vicinal diamine to a triazole moiety mediated by nitric oxide (NO) under aerobic conditions. b, A family of reaction-based dyes that all operate using the same general reaction-based switch as in a. c, Conversion of an anthracene derivative to the corresponding endoperoxide by singlet oxygen cycloaddition.

Figure 3. Representative oxidative cleavage reactions for small-molecule detection

a, Oxidative cleavage of a boronic ester to a phenol triggered by hydrogen peroxide (H₂O₂). b, A family of reaction-based dyes that all operate using the same general reaction-based switch as in part a. c, Dicarbonyl fragmentation assisted by H₂O₂ oxidation. d, Trifluoroketone oxidation and elimination by peroxynitrite. e, Oxidation and cleavage of a p-methoxyphenol by hypochlorous acid (HOCl). f, Oxidative O-dearylation processes mediated by highly reactive oxygen species such as HOCl. g, Ozonolysis of olefins with β-elimination.

Figure 4. Representative reductive cleavage, nucleophilic reactions and tandem processes for detection of small molecules

a, Selective reduction of aromatic azides to anilines by hydrogen sulfide (H₂S). b, A family of reaction-based dyes that all operate using the same general reaction-based switch as in part a. c, Detection of H₂S by a tandem Michael addition. d, Dual nucleophilic addition strategy for H₂S visualization. e, Thiol-mediated cleavage of electron-poor aryl sulfonates. f, Reversible sensing of thiols by disulfide–thiol exchange chemistry.

Figure 5. Representative metal–ligand substitution and metal-mediated redox addition/cleavage reactions for small-molecule detection

a, Direct NO detection by displacement of quenched dye–ligand conjugates from metal complexes such as ruthenium tetraphenylporphyrin. b, Phosphate detection through coordination to dizinc cores with dye displacement and/or hydrolysis. c, Displacement of a dye-quenching metal centre induced by H₂S with concomitant precipitation of the metal sulfide. d, Metal-mediated reductive N-nitrosylation of a metal–dye complex for NO detection. e,f, Iron-mediated oxidation and cleavage from pendant dyes induced by H₂O₂.

Figure 6. Representative Lewis acid hydrolysis, organometallic and small-molecule activation reactions for specific metal ion detection

a, Mercury-mediated desulfurization/hydroylsis of thiocarbonyl compounds. b, Copper-catalysed hydrolysis of hydrazines with spirodye ring-opening. c, Mercury-catalysed cycloaddition of thiourea substrates. d, Claisen rearrangement reactions for palladium and platinum detection. e, Oxymercuration of terminal alkynes. f,g, Aerobic copper- or cobalt-mediated C–O bond cleavage.

Figure 7. Representative bioimaging applications with reaction-based small-molecule fluorescent probes for highly reactive species and metal ions

a, Levels of NO in a rat kidney visualized with DAC-P. b, Mitochondrial-localized H₂O₂ fluxes in cancer cells imaged by MitoPY1. c, Detection of H₂S in mammalian cells with SFP-2. d, Visualizing accumulation of mercury pools in zebrafish with a spirorhodamine-based probe. e, Production of NO in olfactory bulb slices on depolarization detected by the metal–dye probe Cu2(FL2E). f, Visualization of myeloperoxidase-derived HOCl in a mouse model for peritonitis with SNAPF. Figures reproduced with permission from: a, ref. 10 © 2005 ACS; c, ref. 52 © 2011 NPG; d, ref. 84 © 2006 ACS; e, ref. 75 © 2010 PNAS; f, ref. 44 © 2007 Elsevier; b was kindly provided by B. C. Dickinson.